The objective of Project 3 is to genetically manipulate cancer cells to increase intrinsic radiation sensitivity preferentially to tumor tissue with the ultimate goal of improving the outcome of radiation therapy. To achieve this goal, we will conduct a series of Phase I and II clinical trials that will test the general hypothesis that combining replication-competent adenovirus-mediated double suicide gene therapy with conformal radiotherapy can be applied safely in humans and will demonstrate superior efficacy compared to conformal radiotherapy alone. Specific Aim 1 describes a Phase I/II trial to determine whether replication-competent adenovirus-mediated double suicide gene therapy in combination with conventional dose (72 Gy) intensity modulated radiotherapy (IMRT) is superior to IMRT alone in patients with newly diagnosed, intermediate-to-high risk prostate cancer. The best second-generation adenovirus developed in Project 1 will be used. The primary endpoint will be local tumor control as determined by prostate biopsy status at two years. Other endpoints will be acute and late toxicity, early tumor control at six months and one year, and freedom from biochemical or clinical failure. Specific Aim 2 describes a Phase I/II trial to determine the safety and efficacy of replication-competent adenovirus-mediated double suicide gene therapy in combination with salvage IMRT in patients with locally recurrent prostate cancer. The best second-generation adenovirus developed in Project 1 will be used. Three cohorts of three to six patients will receive a single intraprostatic injection of adenovirus (10[12] vp) along with three weeks of 5-FC + vGCV prodrug therapy and an escalating dose (20, 26, 30 Gy) of IMRT. If there are no toxicity concerns at six months, a Phase II trial will be conducted in which patients will receive a single intraprostatic injection of adenovirus (10[12] vp) along with three weeks of 5-FC + vGCV prodrug therapy and the maximum tolerated dose (MTD) of IMRT. The primary endpoint will be local tumor control as determined by prostate biopsy status at two years. Other endpoints will be acute and late toxicity, and freedom from biochemical or clinical failure. Specific Aim 3 describes a Phase I trial that will evaluate the efficiency of therapeutic gene transfer in vivo using an improved vector formulation designed to enhance gene delivery. The efficiency of gene transfer and distribution of vector will be evaluated in patients with localized prostate cancer who are scheduled to undergo prostatectomy. These studies will generate important new knowledge that will provide the scientific basis for which future large-scale human trials will be based and may ultimately lead to better cancer treatments.